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United States Patent |
5,665,860
|
Pettit
,   et al.
|
September 9, 1997
|
Human cancer inhibitory peptide amides
Abstract
This application discloses seven newly synthesized pentapeptide amides and
our tetrapeptide amides. The synthesis utilized both naturally occurring
and modified amino acids; the modified amino acids are constituents of the
well known dolastatin 10 and dolastatin 15 which are structurally distinct
peptides with excellent antineoplastic activity. These peptides were
constructed by introducing a peptide bond between selected amino acids and
modified amino acids and coupling the resulting di- and tri-peptides to
obtain peptides having a high anticancer activity against a series of
human cancer cell lines.
##STR1##
Inventors:
|
Pettit; George R. (Paradise Valley, AZ);
Srirangam; Jayaram K. (Tempe, AZ);
Williams; Michael D. (Mesa, AZ)
|
Assignee:
|
Arizona Board of Regents acting on behalf of Arizona State University (Tempe, AZ)
|
Appl. No.:
|
671121 |
Filed:
|
June 13, 1996 |
Current U.S. Class: |
530/330; 530/321 |
Intern'l Class: |
A61K 038/04; C07K 005/00 |
Field of Search: |
530/330,321
514/18
|
References Cited
U.S. Patent Documents
4816444 | Mar., 1989 | Pettit et al. | 514/17.
|
5410024 | Apr., 1995 | Pettit et al. | 530/330.
|
5530097 | Jun., 1996 | Pettit et al. | 530/330.
|
Primary Examiner: Hutzell; Paula K.
Assistant Examiner: Prickril; Benet
Attorney, Agent or Firm: Mybeck; Richard R.
Goverment Interests
Financial assistance for this project was provided by U.S. Government Grant
Number OIG-CA44344-01-04-05; and the United States Government may own
certain rights to this invention.
Parent Case Text
This application is a continuation of U.S. patent application, Ser. No.
08/283,684 filed on Aug. 1, 1994, now U.S. Pat. No. 5,530,097.
Claims
Accordingly, what is claimed is:
1. A compound having the general structure below:
##STR4##
wherein R is selected from the group consisting of: Doe "10a"; NH-2ClPEA
"10b"; NH-3ClPEA, "10c"; and NH-4ClPEA, "10d".
2. A compound according to claim 1 wherein R=Doe.
3. A compound according to claim 1 wherein R=NH-2ClPEA.
4. A compound according to claim 1 wherein R=NH-3ClPEA.
5. A compound according to claim 1 wherein R=NH-4ClPEA.
Description
INTRODUCTION
This invention relates generally to the field of cancer chemotherapy and
more particularly to the synthesis of unique pentapeptide amides and
tetrapeptide amide derivatives of dolastatin 10 which may be useful in
chemotherapy.
BACKGROUND OF THE INVENTION
Ancient marine invertebrate species of the Phyla Bryozoa, Molluska, and
Porifera have been well established in the oceans for over one billion
years. Such organisms have undergone trillions of biosynthetic reactions
of their evolutionary chemistry to reach their present level of cellular
organization, regulation and defense.
For example, marine sponges have changed minimally in physical appearance
for nearly 500 million years. This suggests a very effective chemical
resistance to evolution in response to changing environmental conditions
over that period of time. Recognition of the potential for utilizing this
biologically potent marine animal for medicinal purposes was recorded in
Egypt about 2,700 B.C. and by 200 B.C. certain sea hare extracts were
being used in Greece for their curative affect. This consideration along
with the observation that marine animals, e.g. invertebrates and sharks,
rarely develop cancer led to the systematic investigation of marine animal
and plant anticancer compounds.
By 1968, ample evidence had been obtained, based on the U.S. National
Cancer Institute's (NCI) key experimental cancer study systems, that
certain marine organisms could provide new and antineoplastic and/or
cytotoxic agents useful in chemotherapy and might also lead to compounds
which would be effective in the control and/or eradication of viral
diseases.
Further, these marine organisms were believed to possess potentially useful
drug candidates of unprecedented structure which had eluded discovery by
other methods of medicinal chemistry. Fortunately, these expectations have
been realized, e.g. the discovery of the bryostatins, dolastatins and
cephalostatins, many of which are now in preclinical development or human
clinical studies.
Those researchers presently involved in medicinal chemistry know well the
time lag between the isolation of a new compound and its introduction to
the market. Often this procedure takes several years and may take decades.
As a result, industry, in association with the U.S. Government, has
developed a system of testing criteria which serves two purposes. One is
to eliminate those substances which are shown through testing to be
economically counterproductive to pursue. The second, more important
purpose serves to identify those compounds which demonstrate a high
likelihood of success and therefore warrant the further study and
qualification, and attendant expense, necessary to meet the stringent
regulatory requirements which control the ultimate market place.
The current cost to develop the necessary data required for lawful
marketing of a new drug compound approaches ten million dollars per
compound. Economics dictate that such a huge investment be made only when
there is a reasonable likelihood that it can be recovered. Absent such a
likelihood, there will be no investment and, without investment, the
research requisite for the discovery of these potentially life saving
compounds will cease.
Current research in the control of cancer in the United States is
coordinated by the National Cancer Institute (NCI). To determine whether a
substance has anti-cancer properties, the NCI has established a systematic
protocol. This protocol, which involves the testing of a substance against
a standard cell line panel containing 60 human tumor cell lines, has been
verified and is accepted in scientific circles. The protocol, and the
established statistical means for analyzing the results obtained by the
standardized testing are fully described in the literature. See: Boyd, Dr.
Michael R., Principles & Practice of Oncology, PPO Updates, Volume 3,
Number 10, October 1989, for an in depth description of the testing
protocol; and Paull, K. D., "Display and Analysis of Patterns of
Differential Activity of Drugs Against Human Tumor Cell Lines; Development
of Mean Graph and COMPARE Algorithm", Journal of the National Cancer
Institute Reports, Vol. 81, No. 14, Page 1088, Jul. 14, 1989 for a
description of the methods of statistical analysis. Both of these
references are incorporated herein by this reference thereto.
Numerous substances have been discovered which demonstrate significant
antineoplastic or tumor inhibiting characteristics. As stated above, many
of these compounds have been extracted, albeit with great difficulty, from
marine animals such as the sponge and sea hare. Once isolation and testing
of these compounds has been accomplished, a practical question remains,
namely how to produce commercially significant quantities of the desired
substance.
Quinine, which is available in practical quantities from the bark of the
cinchona plant, differs from the compounds which are extracts of marine
creatures possessing antineoplastic qualities. The collection and
processing of these later compounds from their natural sources ranges from
grossly impractical to the utterly impossible. Ignoring the ecological
impact, the population of these creatures and the cost of collection and
extraction make the process unworkable. Artificial synthesis of the active
compounds is the only possible solution.
Therefore, the elucidation of the structure of these antineoplastic
compounds is essential. After the structure has been determined, then a
means of synthesis must be determined. This is often a long and arduous
procedure due to the idiosyncratic complexity of these naturally
occurring, evolutionary modified compounds. In addition, research is
necessary to determine whether any portion of the naturally occurring
compound is irrelevant to the desired properties, so that focus can be on
the simplest structure having the perceived properties.
The Constitution of the United States (Art. 1, Sec. 8) authorized Congress
to establish the United Staes Patent and Trademark office (USPTO) to
promote scientific progress. In order to obtain patent rights, one must
show the utility of the invention. Cancer cell growth in humans often
causes pain, suffering, and premature death. The inhibition of human
cancerous tumor growth as evidenced by NCI cell line data is utilitarian
in that it relieves these conditions, thereby allowing the human thus
afflicted to have a longer, more productive life. Little could be more
utilitarian than this result.
The sole right obtained from the grant of a Letters Patent is to prevent
others from exploiting the subject matter of the patent. This results in
the protection of the inventor for a period adequate to allow the
recoupment of investment. This in turn provides incentive and the means
for further research.
The recognition of antineoplastic and tumor inhibiting activity as
demonstrated by accepted NCI criteria as "utility" can promote research
efforts in the United States and is unequivocally essential if those
efforts are to obtain even a modest modicum of success. To reject the NCI
criteria on any grounds can only result in quashing all further efforts in
the United States and leave our people at the mercy of those foreign
companies who operate in more foresighted jurisdictions.
BRIEF SUMMARY OF THE INVENTION
The investigation of potentially useful antineoplastic peptides offers one
of the most promising approaches to new anticancer drugs. Continuing
research along these lines has now resulted in the discovery and synthesis
of seven new pentapeptide amides and four new tetrapeptide amides. In the
syntheses of these peptides, naturally occurring as well as some modified
amino acids have been utilized. The modified amino acids disclosed herein
are constituents of the well known dolastatin 10 and dolastatin 15 which
are structurally distinct peptides with excellent antineoplastic activity.
Presently dolastatin 10 represents the most important member of the
dolastatin family and is a potentially useful anticancer drug. Herein
disclosed are new compounds having excellent activity against a series of
human cancer cell lines.
The novel peptides disclosed herein were constructed by introduction of a
peptide bond between selected amino acids and modified amino acids and
coupling the resulting di- and tri-peptides to obtain peptides having a
very high anticancer activity. The research has led to the discovery and
synthesis of new and very potent anticancer peptide amides. The present
disclosure involves eleven such compounds: namely seven pentapeptide
amides herein designated 8a-g and four tetrapeptide amides herein
designated 10a-d.
The synthesis of these compounds was achieved in the following manner using
the following terminology and abbreviations: the abbreviations are as
follows:
##STR2##
The common tripeptide 5 needed for the synthesis of these peptides was
synthesized starting from dolaisoleuine (Dil), a modified amino acid.
Dolaisoleuine was coupled with N-cbz-(L)-Leucine (1) using bromotris
(dimethylamino) phosphonium hexafluorophosphate (BrOP) as the coupling
agent in presence of diisopropylethylamine to obtain the dipeptide
N-Z-Leu-Dil-OBu.sup.t (3). The N-carbobenzyloxy protecting group of the
dipeptide 3 was then removed with 5% Pd--C in cyclohexene to afford the
free base which was coupled with dolavaline (Dov, a modified amino acid)
with diethyl cyanophosphonate (DECP) as the coupling agent to give the
required tripeptide Dov-Leu-Dil-OBu.sup.t (5). The t-boc protecting group
of the tripeptide (5) was then removed with trifluoroacetic acid to obtain
the trifluoroacetate salt (6).
The resulting tripeptide-tfa salt (6) was coupled with seven known
dipeptide amide trifluoroacetate salts (7a-g) as well as four known
dolaproine amide trifluoroacetate salts (9a-d) using DECP as the coupling
agent to obtain the respective pentapeptide amides (8a-g) and tetrapeptide
amides (10a-d) in good yields.
All these compounds demonstrated excellent growth inhibition when
administered to a variety of human cancer and mouse leukemia cell lines.
The biological results are disclosed in Tables 1 and 2 below.
Accordingly, the primary object of the subject invention is the synthesis
of peptide derivatives of dolastatin 10, which demonstrates extraordinary
inhibition of cell growth and/or anticancer activity at substantially
reduced cost.
Another object of the present invention is to identify the active portions
of dolastatin 10 derivatives which can be attached to other molecules to
provide an equally effective but considerably less expensive tumor
inhibiting agents.
These and still further objects as shall hereinafter appear are readily
fulfilled by the present invention in a remarkably unexpected manner as
will be readily discerned from the following detailed description of
exemplary embodiments thereof.
DESCRIPTION OF THE PREFERRED EMBODIMENT
In vitro testing is an absolutely essential factor in the ongoing venture
to discover new compounds for use in fighting the scourge of cancer.
Without such screening, the process of obtaining new candidate drugs would
be even more complex and expensive, if not impossible. To understand this
process, and recognize the outstanding results demonstrated by some of the
compositions disclosed herein, one must first understand the procedures,
the nomenclature, and the data analysis involved. A brief description of
the appropriate terminology follows:
ED.sub.50 (P388) and GI.sub.50 (HTCL) identify the drug dose which reduces
the percent tumor/cell growth to 50%. There is no mathematical difference
between ED.sub.50 and GI.sub.50, both of which are calculated using the
same formula. The only difference is historical usage.
TGI, means "Total Growth Inhibition", and identifies the drug dose needed
to yield zero percent growth, i.e. there are just as many cells at the end
of the experiment as were present at the beginning. Whether just as many
cells were killed as were produced (steady state), or no growth occurred
(total inhibition) cannot be distinguished.
LC.sub.50, means "Lethal Concentration 50%", and identifies the drug
concentration which reduces to one-half of the cells originally present at
the beginning of the experiment.
Each drug is tested at five (5) doses: 100-10-1-0.1-0.01-.mu.g/mL. Percent
Growths are calculated for each dose. The two (or three) doses with growth
values above, below, (or near to) 50% growth are used to calculate the
ED.sub.50 /GI.sub.50 values using a linear regression computation. If no
dose yields a growth value under 50%, the results are expressed as:
ED.sub.50 >(highest dose). If no dose yields growth higher than 50%
growth, then ED.sub.50 <(lowest dose). Similar calculations are performed
for the TGI at 0% growth, and at -50% growth for the LC.sub.50.
At the start of each experiment, cells from the in vitro cell cultures are
inoculated into the appropriate tubes or microtiter plates. One set of
control tubes/plates is immediately counted to determine the number of
cells at the start of the experiment. This is the "baseline count", or
"Tzero reading". At the end of the experiment (48 hrs later), a second set
of control tubes/plates is analyzed to determine the "Control Growth"
value. The growth (or death) of cells relative to the initial quantity of
cells is used to define the "Percent of
______________________________________
EXAMPLE:
Baseline Count 20
Control Count 200
(10-Fold Growth)
100% Growth = Control Growth
100% Growth = 200
##STR3## 50% Growth = 110
0% Growth = Tzero 0% Growth = 20
-50% Growth = Tzero/2 -50% Growth = 10
______________________________________
Now that the relevant definitions and data analysis techniques have been
disclosed, this disclosure can now turn to the particular compounds
disclosed herein.
The synthesis of potentially useful peptides presents one of the most
essential and promising approaches to new types of anticancer and
immunosuppressant drugs. The Dolastatins, an unprecedented series of
linear and cyclic antineoplastic and/or cytostatic peptides isolated from
Indian Ocean sea hare Dolabella auricularia represent excellent leads for
synthetic modification. The very productive sea hare Dolabella auricularia
has produced a number of structurally distinct peptides with excellent
antineoplastic activity. Presently Dolastatin 10, a linear pentapeptide,
represents the most important member and is a potentially useful
antineoplastic agent. Dolastatin 10 shows one of the best antineoplastic
activity profiles against various cancer screens presently known. Recently
the total synthesis and absolute configuration of this structurally unique
and biologically active peptide was discovered. This compound has been
tested in vivo and demonstrated significant activity, as shown below.
______________________________________
Experimental Anticancer Activity of Dolastatin 10 in
Murine in vivo Systems, T/C (.mu.g/kg)
______________________________________
P388 Lymphocytic Leukemia
B16 Melanoma
toxic (13.0) 238 and 40% cures (11.11)
155 and 17% cures (6.5)
182 (6.67)
146 and 17% cures (3.25)
205 (4.0)
137 (1.63) 171 (3.4)
L1210 Lymphocytic Leukemia
142 (1.44)
152 (13) M5076 Ovary Sarcoma
135 (6.5) toxic (26)
139 (3.25) 166 (13)
120 (1.63) 142 (6.5)
Human Mammary Xenograph
151 (3.25)
Nude Mouse LOX Human Melanoma
Toxic (26) Xenograph (Nude Mouse)
137 (13) toxic (52)
178 (6.25) 301 and 67% cures (26)
OVCAR-3 Human Ovary Xenograph
301 and 50% cures (13)
Nude Mouse 206 and 33% cures (6.5)
300 (40) 170 and 17% cures (3.25)
MX-1 Human Mammary Xenograft
LOX in separate experiments
(Tumor Regression) 340 and 50% cures (43)
14 (52) 181 and 33% cures (26)
50 (26) 192 (15)
61 (13) 138 and 17% cures (9.0)
69 (6.25)
______________________________________
Dolastatin 10 has also been tested against a minipanel from the NCI Primary
screen. These results appear below, showing the amount of Dolastatin 10
required to attain GI.sub.50 in .mu.g/ml, against the cell lines set forth
below.
##EQU1##
Similarly, the compounds disclosed herein have also been tested against an
NCI in vitro mini panel. For each of six cell lines GI.sub.50, TGI, and
LC.sub.50 amounts were calculated for each compound. Each compound was
also tested against the PS-388 cell line and for this test an ED.sub.50
was calculated.
The protocols followed, for the NCI minipanel are, except for the number of
cell lines, those established by M. R. Boyd Ph.D., and well known to those
of ordinary skill in the art. The procedure followed for the test against
PS-388 Leukemia is the same that was followed in the superseded NCI P-388
screening test, which is also well known to those having ordinary skill in
the art.
TABLE 1
__________________________________________________________________________
Human Cancer-Cell line and PS-388 Mouse Leukemia (ED.sub.50) data for the
pentapeptide amides 8a-g
Cell type
Cell line
8a 8b 8c 8d 8e 8f 8g
__________________________________________________________________________
GI-50 (.mu.g/ml)
Ovarian
OVCAR-3
0.00047
0.000031
0.000064
0.0000085
0.00008
0.0033
>0.000006
CNS SF-295
0.0034
0.0029
0.00088
0.00034
0.0036
>0.01
0.00041
Renal A498 0.0037
0.00045
0.00042
0.00018
0.00087
>0.01
0.00026
Lung-NSC
NCI-H460
0.0028
0.003
0.0017
0.00032
0.0032
>0.01
0.00032
Colon KM20L2
0.0018
0.00028
0.00021
0.000015
0.00034
0.0074
0.000019
Melanoma
SK-MEL-5
0.042
0.000018
0.000042
0.0000051
0.000051
0.0043
>0.000006
TGI (.mu.g/ml)
Ovarian
OVCAR-3
0.005
0.0016
0.0011
0.00023
0.0029
>0.01
>0.001
CNS SF-295
>0.01
>0.01
>0.01
>0.01
>0.01
>0.01
>0.01
Renal A498 >0.01
>0.01
>0.01
0.0038
>0.01
>0.01
>0.01
Lung-NSC
NCI-H460
0.0079
0.009
0.0054
0.0011
>0.01
>0.01
0.0012
Colon KM20L2
>0.01
>0.01
0.0012
0.0012
>0.01
>0.01
0.0012
Melanoma
SK-MEL-5
>0.01
>0.01
0.000174
>0.01
>0.01
>0.01
>0.01
LC-50 (.mu.g/ml)
Ovarian
OVCAR-3
>0.01
>0.01
>0.01
>0.01
>0.01
>0.01
>0.01
CNS SF-295
>0.01
>0.01
>0.01
>0.01
>0.01
>0.01
>0.01
Renal A498 >0.01
>0.01
>0.01
>0.01
>0.01
>0.01
>0.01
Lung-NSC
NCI-H460
>0.01
>0.01
>0.01
>0.01
>0.01
>0.01
>0.01
Colon KM20L2
>0.01
>0.01
>0.01
>0.01
>0.01
>0.01
>0.01
Melanoma
SK-MEL-5
>0.01
>0.01
>0.01
>0.01
>0.01
>0.01
>0.01
ED-50 (.mu.g/ml)
Mouse PS-388
0.00256
0.000434
0.0000371
0.0000025
0.000839
0.00405
0.000271
Leukemia
__________________________________________________________________________
TABLE 2
__________________________________________________________________________
Human Cancer-Cell line and PS-388 Mouse Leukemia (ED.sub.50)
data for the tetrapeptide amides 10a-d
Cell type
Cell line
10a 10b 10c 10d
__________________________________________________________________________
GI-50 (.mu.g/ml)
Ovarian
OVCAR-3
0.000031
0.00031
0.0019
0.00029
CNS SF-295
0.00027
0.00042
0.0035
0.0017
Renal A498 0.00026
0.00069
0.0038
0.0059
Lung-NSC
NCI-H460
0.00022
0.00033
0.0029
0.0024
Colon KM20L2
0.000034
0.00031
0.0020
0.00011
Melanoma
SK-MEL-5
0.000058
0.00043
0.0025
0.00091
TGI (.mu.g/ml)
Ovarian
OVCAR-3
0.00051
0.0012
>0.01
0.012
CNS SF-295
>0.01
>0.01
>0.01
0.1
Renal A498 0.0063
>0.01
>0.01
0.29
Lung-NSC
NCI-H460
0.0007
0.0014
0.0084
0.0097
Colon KM20L2
0.001
0.0013
>0.01
0.011
Melanoma
SK-MEL-5
>0.01
>0.01
>0.01
>1
LC-50 (.mu.g/ml)
Ovarian
OVCAR-3
>0.01
>0.01
>0.01
>1
CNS SF-295
>0.01
>0.01
>0.01
>1
Renal A498 >0.01
>0.01
>0.01
>1
Lung-NSC
NCI-H460
>0.01
>0.01
>0.01
>1
Colon KM20L2
>0.01
>0.01
>0.01
>1
Melanoma
SK-MEL-5
>0.01
>0.01
>0.01
>1
ED-50 (.mu.g/ml)
Mouse PS-388
0.0000312
0.000357
0.00314
0.00586
Leukemia
__________________________________________________________________________
The compound identified by reference (3) N-Z-Leu-Dil-OBu.sup.t was prepared
in the following manner, following what is identified below as General
Procedure A.
General Procedure A
To a solution of the hydrochloride salt of Dolaisoleuine t-butyl ester(2,
4.39 mM) and N-Z-(L)-Leucine (1, 4.83 mM) in dry dichloromethane(15 mL),
cooled to ice-bath temperature (0.degree.-5.degree. C.) was added
diisopropylethylamine(14.49 mM) followed by BrOP(4.83 mM) and the
resulting solution was stirred at the same temperature for 2 hours. The
solvents were removed under reduced pressure and the residue was
chromatographed on a SILICA GEL column using 1:4 acetone-hexane as the
solvent to obtain the required dipeptide as an oily substance (3, 72%);
R.sub.f 0.53 (1:4 acetone-hexane); [.alpha.].sub.D.sup.25 -33.4.degree. (c
6.2, CH.sub.3 OH); IR(neat): 2961, 1723, 1640, 1528, 1456, 1368, 1254,
1154 and 1101 cm.sup.-1 ; .sup.1 H NMR(CDCl.sub.3, 300 MHz): 7.32(m, 5H,
ArH), 5.47 (d, J=8.9 Hz, 1H, NH), 5.08(s, 2H, ArCH.sub.2), 4.68(m, 1H,
dil N--CH), 4.55(m, 1H, Leu C.sup..alpha. H), 3.87(m, 1H, C H--OMe),
3.32(s, 3H, OMe), 2.92(s, 3H, N--Me), 2.26-2.46(m, 2H, CH.sub.2 CO) ,
1.30-1.70(m, 6H, 2.times.CH.sub.2, 2.times.CH), 1.44, 1.43(s, 9H, t-Bu)
and 0.80-1.04(m, 12H, 4.times.CH.sub.3); EIMS (m): 506(M.sup.+), 348, 279,
220, 177, 128, 100(100%) and 91.
The compound identified by reference (5) Dov-Leu-Dil-OBu.sup.t was prepared
in the following manner, following what is identified below as General
Procedure B.
General Procedure B
A solution of Z-Leu-Dil-OBu.sup.t (3, 2.22 mM) was dissolved in anhydrous
methanol (10 mL) and cyclohexene (10 mL) was added in a nitrogen
atmosphere. To the solution was added 5% Pd--C (1.15 g) and the mixture
was heated at reflux for 6 minutes. The catalyst was removed by filtering
through a layer of celite, the solvent removed under reduced pressure, and
the residue dried in high vacuum for 2 hours.
To a solution of the above free base and N,N-dimethyl- (L)-valine(4, 2.66
mM) in dry dichloromethane (10 mL) was added triethylamine (2.66 mM)
followed by DECP (2.66 mM) at 0.degree.-5.degree. C. under argon
atmosphere. After stirring at the same temperature for 2 hours, the
solvent was removed and the residue chromatographed on a SILICA GEL column
with 15% acetone in hexane as solvent to give the required tripeptide
t-butyl ester as a colorless gummy mass (5, 65%); R.sub.f 0.69 (30%
acetone-hexane); [.alpha.].sub.d.sup.25 -24.8.degree. (c 5.0, CH.sub.3
OH); IR(neat): 2961, 1730, 1626, 1524, 1452, 1368, 1294, 1154 and 1101
cm.sup.-1 ; .sup.1 H NMR(CDCl.sub.3, 300 MHz): 6.82(br d, J=8.8 Hz, 1H,
NH), 5.01(m, 1H, dil C HN), 4.60(br m, 1H, Leu C.sup..alpha. --H), 3.85
(m, 1H, C H--OMe), 3.33(s, 3H, OMe), 2.97(s, 3H, dil N--Me), 2.2-2.5(m,
2H, CH.sub.2 --CO), 2.24(s, 6H, NMe.sub.2), 2.05(m, 1H, dov C.sup..alpha.
--H), 1.2-1.8(m, 7H, 2.times.CH.sub.2, 3.times.CH), 1.42, 1.44(s, 9H,
t-Bu) and 0.75-0.99(m, 18H, 6.times.CH.sub.3); EIMS (m): 499(M.sup.+),
456, 241, 186, 101, and 100(100%).
The tripeptide trifluoroacetate salt, identified above by reference (6) was
synthesized following what is identified below as General Procedure C.
General Procedure C
To a solution of the tripeptide t-butyl ester(5, 10 mM) in
dichloromethane(10 mL) cooled to ice-bath temperature was added
trifluoroacetic acid(10 mL) under argon atmosphere and the solution was
stirred at the same temperature for 1 hour. The solvents were then removed
under reduced pressure, the residue was dissolved in toluene and solvent
again removed under reduced pressure. The residue was dried in vacuo and
crystallized from diethyl ether to obtain the tripeptide trifluoroacetate
salt(6, quantitative) as a colorless solid; M.p. 168.degree.-169.degree.
C.; [.alpha.].sub.D.sup.25 -36.degree. (c 0.1, CHCl.sub.3); IR(thin film):
2938, 2880, 2834, 1672, 1632, 1549, 1485, 1466, 1416, 1385, 1317, 1296,
1240, 1201, 1181, 1136, 1099, 1009, 990, 833, 799, 737, 721 and 617
cm.sup.-1.
The pentapeptide amides herein identified by references 8a-g and 10a-d,
were synthesized following General Procedure D shown below;
General Procedure D
To a solution of the trifluoroacetate salt (7a-g, 9a-d, 0.2 mM) in
methylene chloride (2 mL, distilled from calcium hydride) was added the
Dov-Leu-Dil tripeptide trifluoroacetate salt (6, 0.2 mM) followed by
triethylamine (0.63 mM) and DECP (0.22 mM, ice bath). The solution was
stirred under argon at 0.degree.-5.degree. C. for 1-2 hours. The solvent
was removed (under vacuum at room temperature) and the residue was
chromatographed on a SILICA GEL (0.040-0.063 mm) column. After the
evaporation of solvent from the fractions (selected by thin layer
chromatography) the required peptide amides were obtained as a fluffy
solid.
Chromatography over SILICA GEL with hexane-acetone (2:3) as eluent,
according to General Procedure D, yielded
L-Dolavalyl-L-Leucyl-N-methyl-(3R,4S,5S)-Dolaisoleuinyl-(2R,3R,4S)-Dolapro
inyl-L-Methionine N-2-chlorophenylamide (8a) as a white solid (C.sub.43
H.sub.73 N.sub.6 O.sub.7 S.sub.1 Cl.sub.1, 81); Rf 0.26 (hexane-acetone
1:1); M. p. 88.degree.-90.degree. C., [.alpha.].sub.D.sup.23
=-57.6.degree. (c 0.17, CHCl.sub.3); IR (thin film): 3293, 2963, 2932,
2876, 1628, 1593, 1532, 1441, 1385, 1370, 1294, 1269, 1233, 1200, 1165,
1134, 1099, 1051 and 752 cm.sup.-1 ; EIMS (70 eV) m: 852 (M.sup.+).
Chromatography over SILICA GEL with hexane-acetone (1:1) as eluent
according to General Procedure D, yielded
L-Dolavalyl-L-Leucyl-N-methyl-(3R,4S,5S)-Dolaisoleuinyl-(2R,3R,4S)-Dolapro
inyl-L-Methionine N-4-chlorophenylamide (8b) (C.sub.43 H.sub.73 N.sub.6
O.sub.7 S.sub.1 Cl.sub.1, 98%); Rf 0.32 (hexane-acetone 1:1); M. p.
95.degree.-96.degree.C.; [.alpha.].sub.D.sup.23 =-64.4.degree. (c 0.09,
CHCl.sub.3); IR (thin film): 3306, 3293, 2961, 2934, 2874, 1643, 1626,
1543, 1493, 1449, 1418, 1404, 1385, 1368, 1304, 1289, 1269, 1250, 1198,
1169, 1134, 1098, 1038 and 829 cm.sup.-1 ; EIMS (70 eV) m/z: 852
(M.sup.+).
Chromatography over SILICA GEL with hexane-acetone (1:1) as eluent,
according to General Procedure D, yielded
L-Dolavalyl-L-Leucyl-N-methyl-(3R,4S,5S)-Dolaisoleuinyl-(2R,3R,4S)-Dolapro
inyl-L-Phenylalanine N-3-chlorophenylamide (8c) (C.sub.47 H.sub.73 N.sub.6
O.sub.7 Cl.sub.1, 99%); Rf 0.34 (hexane-acetone 1:3); M. p.
86.degree.-88.degree.C.; [.alpha.].sub.D.sup.23 =-47.8.degree. (c 0.18,
CHCl.sub.3); IR (thin film): 3306, 3293, 2963, 2934, 2876, 2832, 1649,
1626, 1595, 1545, 1483, 1452, 1425, 1385, 1368, 1302, 1267, 1250, 1236,
1194, 1167, 1134, 1099, 1038, 978, 779 and 741 cm.sup.-1 ; EIMS (70 eV)
m/z: 868 (M.sup.+).
Chromatography over SILICA GEL with hexane-acetone (2:3) as eluent,
according to General Procedure D, yielded
L-Dolavalyl-L-Leucyl-N-methyl-(3R,4S,5S)-Dolaisoleuinyl-(2R,3R,4S)-Dolapro
inyl-L-Phenylalanine N-4-chlorophenylamide (8d) as a glassy solid (C.sub.47
H.sub.73 N.sub.6 O.sub.7 Cl.sub.1, 82%); Rf 0.33 (hexane-acetone 1:1); M.
p. 88.degree.-90.degree. C.; [.alpha.].sub.D.sup.23 =-54.3.degree. (c
0.14, CHCl.sub.3); IR (thin film): 3306, 3295, 2961, 2932, 2874, 1649,
1626, 1543, 1493, 1454, 1418, 1404, 1385, 1368, 1306, 1290 1269, 1248,
1200, 1134, 1098, 1038, 1015 and 829 cm.sup.-1 ; EIMS (70 eV) m/z: 868
(M.sup.+).
Chromatography over SILICA GEL with acetone-hexane (3:2) as eluent,
according to General Procedure D, yielded
L-Dolavalyl-L-Leucyl-N-methyl-(3R,4S,5S)-Dolaisoleuinyl-(2R,3R,4S)-Dolapro
inyl-L-Methionine N-2-benzothiazolamide (8e) (C.sub.44 H.sub.73 N.sub.7
O.sub.7 S.sub.2, 93%); Rf 0.27 (hexane-acetone 1:1);
[.alpha.].sub.D.sup.25 =-49.2.degree. (c 0.13, CHCl.sub.3); M. p.
90.degree.-92.degree. C.; IR (thin film): 3306, 3293, 3214, 3196, 2961,
2932, 2874, 1626, 1547, 1443, 1420, 1387, 1368, 1263, 1235, 1194, 1165,
1099, 1036 and 756 cm.sup.-1 ; EIMS m/z: 875 (M.sup.+).
Chromatography over SILICA GEL with acetone-hexane (3:1) as eluent,
according to General Procedure D, yielded
L-Dolavalyl-L-Leucyl-N-methyl-(3R,4S,5S)-Dolaisoleuinyl-(2R,3R,4S)-Dolapro
inyl-L-Proline N-2-benzothiazolamide (8f) (C.sub.44 H.sub.71 N.sub.7
O.sub.7 S,60%); Rf 0.20 (hexane-acetone 1:1); [.alpha.].sub.D.sup.25
=39.1.degree. (c 0.11, CHCl.sub.3); M. p. 96.degree.-99.degree. C.; IR
(thin film): 3306, 2961, 2932, 2876, 1703, 1626, 1549, 1443, 1385, 1263,
1169 and 1098 cm.sup.-1 ; EIMS m/z: 842 (M.sup.+).
Chromatography over SILICA GEL with acetone-hexane (3:1) as eluent,
according to General Procedure D, yielded
L-Dolavalyl-L-Leucyl-N-methyl-(3R,4S,5S)-Dolaisoleuinyl-(2R
,3R,4S)-Dolaproinyl-L-Methionine N-3-quinolinamide (8g) as a glassy solid
(C.sub.46 H.sub.75 N.sub.7 O.sub.7 S, 83%); Rf 0.14 (hexane-acetone 1:1);
[.alpha.].sub.D.sup.25 =46.9.degree. (c 0.16, CHCl.sub.3); M. p.
118.degree.-120.degree.C.; IR (thin film) n: 3291, 2963, 2934, 2876, 1649,
1632, 1580, 1555, 1489, 1452, 1422, 1385, 1368, 1346, 1304, 1283, 1271,
1202, 1181, 1134, 1099, 1042, 785, 754, 719 and 615 cm.sup.-1 ; EIMS m/z:
869 (M.sup.+).
Chromatography over SILICA GEL with acetone-hexane (3:1) as eluent,
according to General Procedure D, yielded
Dolavalyl-L-Leucyl-N-methyl-(3R,4S,5S)
-Dolaisoleuinyl-(2R,3R,4S)-Dolaproinyl-Dolaphenine (10a) as a glassy solid
(C.sub.43 H.sub.70 N.sub.6 O.sub.6 S, 84%); M. p. 77.degree.-78.degree.
C.; R.sub.f 0.2 (acetone-hexane 1:1); [.alpha.].sub.D.sup.25 -68.6.degree.
(c 0.14, CHCl.sub.3); IR(thin film): 3295, 2960, 2934, 2876, 2830, 1624,
1535, 1497, 1454, 1418, 1385, 1370, 1319, 12287, 1267, 1225, 1200, 1171,
1136, 1101, 1040, 735, 698, 619, 610 and 536 cm.sup.-1 ; EIMS (m/z):
798(M.sup.+).
Chromatography over SILICA GEL with acetone-hexane (3:1) as eluent,
according to General Procedure D, yielded
L-Dolavalyl-L-Leucyl-N-methyl-(3R,4S,5S)-Dolaisoleuinyl-(2R,3R,4S)-Dolapro
inyl-N-2(2-chlorophenyl) ethylamide (10b) as a gummy mass (C.sub.40
H.sub.68 N.sub.5 O.sub.6 Cl.sub.1, 77%); R.sub.f 0.27 (acetone-hexane
1:1); [.alpha.].sub.D.sup.25 -54.3.degree. (c 0.07, CHCl.sub.3); IR(thin
film): 3308, 2961, 2934, 2876, 2830, 1624, 1537, 1451, 1418, 1383, 1366,
1287, 1269, 1223, 1198, 1169, 1157, 1134, 1101, 1055, 1040 and 754
cm.sup.-1 ; EIMS (m/z): 749(M.sup.+).
Chromatography over SILICA GEL with acetone-hexane (3:1) as eluent,
according to General Procedure D, yielded
L-Dolavalyl-L-Leucyl-N-methyl-(3R,4S,5S)-Dolaisoleuinyl-(2R,3R,4S)-Dolapro
inyl-N-2(3-chlorophenyl) ethylamide (10c) as a gummy mass (C.sub.40
H.sub.68 N.sub.5 O.sub.6 Cl.sub.1, 75%); R.sub.f 0.23 (acetone-hexane
1:1); [.alpha.].sub.D.sup.25 -47.8.degree. (c 0.09, CHCl.sub.3); IR(thin
film): 3308, 2961, 2934, 2876, 2830, 1643, 1624, 1537, 1452, 1418, 1383,
1366, 1289, 1267, 1223, 1200, 1169, 1136, 1101, 1039, 781 and 685
cm.sup.-1 ; EIMS (m/z): 749(M.sup.+).
Chromatography over SILICA GEL with acetone-hexane (3:1) as eluent,
according to General Procedure D, yielded
L-Dolavalyl-L-Leucyl-N-methyl-(3R,4S,5S)-Dolaisoleuinyl-(2R,3R,4S)-Dolapro
inyl-N-2(4-chlorophenyl) ethylamide (10d ) as a glassy solid (C.sub.40
H.sub.68 N.sub.5 O.sub.6 Cl.sub.1, 79%); R.sub.f 0.54 (acetone-hexane
3:1); M. p. 67.degree.-70.degree. C.; [.alpha.].sub.D.sup.25 -72.2.degree.
(c 0.09, CHCl.sub.3); IR(thin film): 3308, 2961, 2934, 2876, 1624, 1541,
1493, 1451, 1418, 1385, 1366, 1269, 1225, 1198, 1136, 1099 and 1040
cm.sup.-1 ; EIMS (m/z): 749(M.sup.+).
To further aid in the understanding of the present invention, and not by
way of limitation the following examples are presented.
EXAMPLE I
N-Z-Leu-Dil-OBu.sup.t (3) was prepared as follows: To a solution of the
hydrochloride salt of Dolaisoleuine t-butyl ester(2, 4.39 mM) and
N-Z-(L)-Leucine (1, 4.83 mM) in dry dichloromethane(15 mL), cooled to
ice-bath temperature (0.degree.-5.degree. C.) was added
diisopropylethylamine(14.49 mM) followed by BrOP(4.83 mM) and the
resulting solution was stirred at the same temperature for 2 hours. The
solvents were removed under reduced pressure and the residue was
chromatographed on a SILICA GEL column using 1:4 acetone-hexane as the
solvent to obtain the required dipeptide as an oily substance (3, 72%);
R.sub.f 0.53 (1:4 acetone-hexane); [.alpha.].sub.D.sup.25 -33.4.degree. (c
6.2, CH.sub.3 OH); IR(neat): 2961, 1723, 1640, 1528, 1456, 1368, 1254,
1154 and 1101 cm.sup.-1 ; .sup.1 H NMR(CDCl.sub.3, 300 MHz): 7.32(m, 5H,
ArH), 5.47 (d, J=8.9 Hz, 1H, NH), 5.08(s, 2H, ArCH.sub.2), 4.68(m, 1H, dil
N--CH), 4.55(m, 1H, Leu C.sup..alpha. H), 3.87(m, 1H, C H--OMe), 3.32(s,
3H, OMe), 2.92(s, 3H, N--Me), 2.26-2.46(m, 2H, CH.sub.2 CO), 1.30-1.70(m,
6H, 2.times.CH.sub.2, 2.times.CH), 1.44, 1.43(s, 9H, t-Bu) and
0.80-1.04(m, 12H, 4.times.CH.sub.3); EIMS (m): 506(M.sup.+), 1348, 279,
220, 177, 128, 100(100%) and 91.
EXAMPLE II
Dov-Leu-Dil-OBu.sup.t (5) was prepared as follows: A solution of
Z-Leu-Dil-OBu.sup.t (3, 2.22 mM) was dissolved in anhydrous methanol (10
mL) and cyclohexene (10 mL) was added in a nitrogen atmosphere. To the
solution was added 5% Pd--C (1.15 g) and the mixture was heated at reflux
for 6 minutes. The catalyst was removed by filtering through a layer of
celite, the solvent removed under reduced pressure, and the residue dried
in high vacuum for 2 hours.
To a solution of the above free base and N,N-dimethyl- (L)-valine(4, 2.66
mM) in dry dichloromethane (10 mL) was added triethylamine (2.66 mM)
followed by DECP (2.66 mM) at 0.degree.-5.degree. C. under argon
atmosphere. After stirring at the same temperature for 2 hours, the
solvent was removed and the residue chromatographed on a SILICA GEL column
with 15% acetone in hexane as solvent to give the required tripeptide
t-butyl ester as a colorless gummy mass (5, 65%); R.sub.f 0.69 (30%
acetone-hexane); [.alpha.].sub.D.sup.25 -24.8.degree. (c 5.0, CH.sub.3
OH); IR(neat): 2961, 1730, 1626, 1524, 1452, 1368, 1294, 1154 and 1101
cm.sup.-1 ; .sup.1 H NMR(CDCl.sub.3, 300 MHz): 6.82(br d, J=8.8 Hz, 1H,
NH), 5.01(m, 1H, dil CHN), 4.60(br m, 1H, Leu C.sup..alpha. --H), 3.85(m,
1H, C H--OMe), 3.33(s, 3H, OMe), 2.97(s, 3H, dil N--Me), 2.2-2.5(m, 2H,
CH.sub.2 --CO), 2.24(s, 6H, NMe.sub.2), 2.05(m, 1H, dov C.sup..alpha.
--H), 1.2-1.8(m, 7H, 2.times.CH.sub.2, 3.times.CH), 1.42, 1.44(s, 9H,
t-Bu) and 0.75-0.99(m, 18H, 6.times.CH.sub.3); EIMS (m): 499(M.sup.+),
456, 241, 186, 101, and 100(100%).
EXAMPLE III
Tripeptide Trifluoroacetate Salt(6) was prepared as follows: To a solution
of the tripeptide t-butyl ester(5, 10 mM) in dichloromethane(10 mL) cooled
to ice-bath temperature was added trifluoroacetic acid(10 mL) under argon
atmosphere and the solution was stirred at the same temperature for 1
hour. The solvents were then removed under reduced pressure, the residue
was dissolved in toluene and solvent again removed under reduced pressure.
The residue was dried in vacuo and crystallized from diethyl ether to
obtain the tripeptide trifluoroacetate salt(6, quantitative) as a
colorless solid; M.p. 168.degree.-169.degree. C.; [.alpha.].sub.D.sup.25
-36.degree. (c 0.1, CHCl.sub.3); IR(thin film): 2938, 2880, 2834, 1672,
1632, 1549, 1485, 1466, 1416, 1385, 1317, 1296, 1240, 1201, 1181, 1136,
1099, 1009, 990, 833, 799, 737, 721 and 617 cm.sup.-1.
EXAMPLE IV
Pentapeptide amides 8a-g, 10a-d were prepared as follows: To a solution of
the trifluoroacetate salt (7a-g, 9a-d, 0.2 mM) in methylene chloride (2
mL, distilled from calcium hydride) was added the Dov-Leu-Dil tripeptide
trifluoroacetate salt (6, 0.2 mM) followed by triethylamine (0.63 mM) and
DECP (0.22 mM, ice bath). The solution was stirred under argon at
0.degree.-5.degree. C. for 1-2 hours. The solvent was removed (under
vacuum at room temperature) and the residue was chromatographed on a
SILICA GEL (0.040-0.063 mm) column. After the evaporation of solvent from
the fractions (selected by thin layer chromatography), the required
peptide amides were obtained as a fluffy solid.
EXAMPLE IV-a
Chromatography over SILICA GEL with hexane-aoetone (2:3) as eluent,
according to General Procedure D, yielded
L-Dolavalyl-L-Leucyl-N-methyl-(3R,4S,5S)-Dolaisoleuinyl-(2R,3R,4S)-Dolapro
inyl-L-Methionine N-2-chlorophenylamide (8a) as a white solid (C.sub.43
H.sub.73 N.sub.6 O.sub.7 S.sub.1 Cl.sub.1, 81%); Rf 0.26 (hexane-acetone
1:1); M. p. 88.degree.-90.degree. C., [.alpha.].sub.D.sup.23 =57.6.degree.
(c 0.17, CHCl.sub.3); IR (thin film): 3293, 2963, 2932, 2876, 1628, 1593,
1532, 1441, 1385, 1370, 1294, 1269, 1233, 1200, 1165, 1134, 1099, 1051 and
752 cm.sup.-1 ; EIMS (70 eV) m/z: 852 (M.sup.+).
EXAMPLE IV-b
Chromatography over SILICA GEL with hexane-acetone (1:1) as eluent,
according to General Procedure D, yielded
L-Dolavalyl-L-Leucyl-N-methyl-(3R,4S,5S)-Dolaisoleuinyl-(2R,3R,4S)-Dolapro
inyl-L-Methionine N-4-chlorophenylamide (8b) (C.sub.43 H.sub.73 N.sub.6
O.sub.7 S.sub.1 Cl.sub.1, 98%); Rf 0.32 (hexane-acetone 1:1); M. p.
95.degree.-96.degree. C.; [.alpha.].sub.D.sup.23 =-64.4.degree. (c 0.09,
CHCl.sub.3); IR (thin film): 3306, 3293, 2961, 2934, 2874, 1643, 1626,
1543, 1493, 1449, 1418, 1404, 1385, 1368, 1304, 1289, 1269, 1250, 1198,
1169, 1134, 1098, 1038 and 829 cm.sup.-1 ; EIMS (70 eV) m/z: 852
(M.sup.+).
EXAMPLE IV-c
Chromatography over SILICA GEL with hexane-acetone (1:1) as eluent,
according to General Procedure D, yielded
L-Dolavalyl-L-Leucyl-N-methyl-(3R,4S,5S)-Dolaisoleuinyl-(2R,3R,4S)-Dolapro
inyl-L-Phenylalanine N-3-chlorophenylamide (8c) (C.sub.47 H.sub.73 N.sub.6
O.sub.7 Cl.sub.1, 99%); Rf 0.34 (hexane-acetone 1:3); M. p.
86.degree.-88.degree. C.; [.alpha.].sub.D.sup.23 =-47.8.degree. (c 0.18,
CHCl.sub.3); IR (thin film): 3306, 3293, 2963, 2934, 2876, 2832, 1649,
1626, 1595, 1545, 1483, 1452, 1425, 1385, 1368, 1302, 1267, 1250, 1236,
1194, 1167, 1134, 1099, 1038, 978, 779 and 741 cm.sup.-1 ; EIMS (70 eV)
m/z: 868 (M.sup.+).
EXAMPLE IV-d
Chromatography over SILICA GEL with hexane-acetone (2:3) as eluent,
according to General Procedure D, yielded
L-Dolavalyl-L-Leucyl-N-methyl-(3R,4S,5S)-Dolaisoleuinyl-(2R,3R,4S)-Dolapro
inyl-L-Phenylalanine N-4-chlorophenylamide (8d) as a glassy solid (C.sub.47
H.sub.73 N.sub.6 O.sub.7 Cl.sub.1, 82%); Rf 0.33 (hexane-acetone 1:1); M.
p. 88.degree.-90.degree. C.; [.alpha.].sub.D.sup.23 =-54.3.degree. (c 0
14, CHCl.sub.3); IR (thin film) n: 3306, 3295, 2961, 2932, 2874, 1649,
1626, 1543, 1493, 1454, 1418, 1404, 1385, 1368, 1306, 1290, 1269, 1248,
1200, 1134, 1098, 1038, 1015 and 829 cm.sup.-1 ; EIMS (70 eV) m/z: 868
(M.sup.+).
EXAMPLE IV-e
Chromatography over SILICA GEL with acetone-hexane (3:2) as eluent
according to General Procedure D, yielded
L-Dolavalyl-L-Leucyl-N-methyl-(3R,4S,5S)-Dolaisoleuinyl-(2R,3R,4S)-Dolapro
inyl-L-Methionine N-2-benzothiazolamide (8e) (C.sub.44 H.sub.73 N.sub.7
O.sub.7 S.sub.2, 93%); Rf 0.27 (hexane-acetone 1:1);
[.alpha.].sub.D.sup.25 =-49.2.degree. (c 0.13, CHCl.sub.3); M. p.
90.degree.-92.degree. C.; IR (thin film): 3306, 3293, 3214, 3196, 2961,
2932, 2874, 1626, 1547, 1443, 1420, 1387, 1368, 1263, 1235, 1194, 1165,
1099, 1036 and 756 cm.sup.-1 ; EIMS m/z: 875 (M.sup.+) .
EXAMPLE IV-f
Chromatography over SILICA GEL with acetone-hexane (3:1) as eluent,
according to General Procedure D, yielded L-Dolavalyl-L-Leucyl
-N-methyl-(3R,4S,5S)-Dolaisoleuinyl-(2R,3R,4S)-Dolaproinyl-L-Proline
N-2-benzothiazolamide (8f) (C.sub.44 H.sub.71 N.sub.7 O.sub.7 S, 60%); Rf
0.20 (hexane-acetone 1:1); [.alpha.].sub.D.sup.25 =-39.1.degree. (c 0.11,
CHCl.sub.3); M. p. 96.degree.-99.degree. C.; IR (thin film): 3306, 2961,
2932, 2876, 1703, 1626, 1549, 1443, 1385, 1263, 1169 and 1098 cm.sup.-1 ;
EIMS m/z: 842 (M.sup.+).
EXAMPLE IV-g
Chromatography over SILICA GEL with acetone-hexane (3:1) as eluent
according to General Procedure D, yielded
L-Dolavalyl-L-Leucyl-N-methyl-(3R,4S,5S)-Dolaisoleuinyl-(2R,3R,4S)-Dolapro
inyl-L-Methionine N-3-quinolinamide (8g) as a glassy solid (C.sub.46
H.sub.75 N.sub.7 O.sub.7 S, 83%); Rf 0.14 (hexane-acetone 1:1);
[.alpha.].sub.D.sup.25 =-46.9.degree. (c 0.16, CHCl.sub.3); M. p.
118.degree.-120.degree. C.; IR (thin film): 3291, 2963, 2934, 2876, 1649,
1632, 1580, 1555, 1489, 1452, 1422, 1385, 1368, 1346, 1304, 1283, 1271,
1202, 1181, 1134, 1099, 1042, 785, 754, 719 and 615 cm.sup.-1 ; EIMS m/z:
869 (M.sup.+).
EXAMPLE V-a
Chromatography over SILICA GEL with acetone-hexane (3:1) as eluent,
according to General Procedure D, yielded
L-Dolavalyl-L-Leucyl-N-methyl-(3R,4S,5S)-Dolaisoleuinyl-(2R,3R,4S)-Dolapro
inyl-Dolaphenine (10a) as a glassy solid (C.sub.43 H.sub.70 N.sub.6 O.sub.6
S, 84%); M.p. 77.degree.-78.degree. C.; R.sub.f 0.2 (acetone-hexane 1.1);
[.alpha.].sub.D.sup.25 -68.6.degree. (c 0.14, CHCl.sub.3); IR(thin film):
3295, 2960, 2934, 2876, 2830, 1624, 1535, 1497, 1454, 1418, 1385, 1370,
1319, 12287, 1267, 1225, 1200, 1171, 1136, 1101, 1040, 735, and 698
cm.sup.-1 ; EIMS (m/z): 798(M.sup.+).
EXAMPLE V-b
Chromatography over SILICA GEL with acetone-hexane (3:1) as eluent
according to General Procedure D, yielded
L-Dolavalyl-L-Leucyl-N-methyl-(3R,4S,5S)-Dolaisoleuinyl-(2R,3R,4S)-Dolapro
inyl-N-2(2-chlorophenyl) ethylamide (10b) as a gummy mass (C.sub.40
H.sub.68 N.sub.5 O.sub.6 Cl.sub.1, 77%); R.sub.f 0.27 (acetone-hexane
1:1); [.alpha.].sub.D.sup.25 -54.3.degree. (c 0.07, CHCl.sub.3); IR(thin
film): 3308, 2961, 2934, 2876, 2830, 1624, 1537, 1451, 1418, 1383, 1366,
1287, 1269, 1223, 1198, 1169, 1157, 1134, 1101, 1055, 1040 and 754
cm.sup.-1 ; EIMS (m/z): 749(M.sup.+).
EXAMPLE V-c
Chromatography over SILICA GEL with acetone-hexane (3:1) as eluent,
according to General Procedure D, yielded
L-Dolavalyl-L-Leucyl-N-methyl-(3R,4R,5S)-Dolaisoleuinyl-(2R,3R,4S)-Dolapro
inyl-N-2(3-chlorophenyl) ethylamide (10c) as a gummy mass (C.sub.40
H.sub.68 N.sub.5 O.sub.6 Cl.sub.1, 75%); R.sub.f 0.23 (acetone-hexane
1:1); [.alpha.].sub.D.sup.25 -47.8.degree. (c 0.09, CHCl.sub.3); IR(thin
film): 3308, 2961, 2934, 2876, 2830, 1643, 1624, 1537, 1452, 1418, 1383,
1366, 1289, 1267, 1223, 1200, 1169, 1136, 1101, 1039, 781 and 685
cm.sup.-1 ; EIMS (m/z): 749(M.sup.+).
EXAMPLE V-d
Chromatography over SILICA GEL with acetone-hexane (3:1) as eluent,
according to General Procedure D, yielded
L-Dolavalyl-L-Leucyl-N-methyl-(3R,4S,5S)-Dolaisoleuinyl-(2R,3R,4S)-Dolapro
inyl-N-2(4-chlorophenyl) ethylamide (10d) as a glassy solid (C.sub.40
H.sub.68 N.sub.5 O.sub.6 Cl.sub.1, 79%); R.sub.f 0.54 (acetone-hexane
3:1); M.p. 67.degree.-70.degree. C.; [.alpha.].sub.D.sup.25 -72.2.degree.
(c 0.09, CHCl.sub.3); IR(thin film): 3308, 2961, 2934, 2876, 1624, 1541,
1493, 1451, 1418, 1385, 1366, 1269, 1225, 1198, 1136, 1099 and 1040
cm.sup.-1 ; EIMS (m/z): 749(M.sup.+).
From the foregoing, it is readily apparent that a useful embodiment of the
present invention has been herein described and illustrated which fulfills
all of the aforestated objectives in a remarkably unexpected fashion. It
is of course understood that such modifications, alterations and
adaptations as may readily occur to the artisan confronted with this
disclosure are intended within the spirit of this disclosure which is
limited only by the scope of the claims appended hereto.
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